Frequent loss of heterozygosity in CRISPR-Cas9-edited early human embryos

Abstract

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4-20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

Keywords: CRISPR-Cas9; genome editing; human embryo; loss of heterozygosity; segmental aneuploidy.

Fig. 1.

Segmental losses/gains of chromosome 6 are prevalent in OCT4-targeted embryo samples. (A) Copy number profile of sample L_C12.02. The segmental gain of chromosome 6 is highlighted. The profile was constructed with 26,000 bins of size 100 kbp, which produced 29 segments. The expected (Eσ) and measured (σ) SD of the profile are reported. (B) Zoomed-in view of the copy number profile for samples with segmental losses or gains of chromosome 6. (C) Zoomed-in view of the copy number profile for samples with normal chromosome 6. The Eσ and σ reported in B and C correspond to the chromosome only. The approximate position of the POU5F1 gene is indicated by a red arrowhead. The red dashed line indicates a copy ratio of 3:2, while the blue dashed lines corresponds to a copy ratio of 1:2. (D) The percentage of control and targeted samples with whole or segmental losses/gains of chromosome 6 according to their copy number profiles. P values are the result of two-tailed Fisher’s tests.

Fig. 2.

LOH in the POU5F1 locus is prevalent among OCT4-targeted embryo samples. (A) SNP profiles constructed from deep sequencing of the depicted amplicons. The four types of LOH events observed are exemplified. Note that there are amplicons with ≥5x coverage in which SNPs were not called because all reads agree with the reference genome. (B) The frequency of each type of LOH event in control and targeted samples. P value is the result of a two-tailed Fisher’s test.

Fig. 3.

LOH in OCT4-targeted samples does not lead to preferential misexpression of genes located on chromosome 6. (A) The fraction of differentially expressed genes per chromosome from the comparison between OCT4-null samples and Cas9 controls. (B) Location of differentially expressed genes along chromosome 6. (C) Volcano plot summarizing the comparison between OCT4-null samples and Cas9 controls with differential gene expression analysis. The chromosome location of some of the most dysregulated genes is shown (absolute log₂ fold change > 20 and Benjamini–Hochberg adjusted P < 0.05). The red dashed lines correspond to absolute log₂ fold changes > 1 and Benjamini–Hochberg adjusted P < 0.05. (D) Genes located on chromosome 6 are not overrepresented in the list of loci whose expression is disturbed upon OCT4 knock out. The same applies for genes directly upstream to the POU5F1 gene. P values are the result of two-tailed Fisher’s tests.

Fig. 4.

Transcriptome-based karyotypes do not capture segmental losses/gains of chromosome 6 in OCT4-targeted embryo samples. (A) Digital karyotype based on the total gene expression deviation from the average of each chromosome arm (z-score-karyotyping). Only chromosome 6 is shown (see SI Appendix, Fig. S14A for the rest of the chromosomes). (B) The percentage of control and targeted samples with segmental losses/gains of chromosome 6 according to their transcriptome-based karyotype (SI Appendix, Figs. S14A and S15). P value is the result of a two-tailed Fisher’s test.